Chiyo Kawaura

Membrane fusion plays an important role in gene transfection mediated by cationic liposomes

By confocal laser scanning microscopy (CLSM) we have studied the membrane fusion between cationic liposomes and the endosome membranes involved in gene transfection mediated by cationic liposomes. Antisense oligonucleotides were transferred by cationic liposomes with a cationic cholesterol derivative, cholesteryl‐3β‐carboxyamidoethylenedimethylamine (I). Cationic liposomes were made by a mixture of the derivative I and DOPE. The intracellular distribution of fluorescein‐conjugated antisense oligonucleotides (phosphorothioate) was studied by CLSM. The images showed that the antisense oligonucleotides were preferentially transferred into the nucleus of target cells (NIH3T3, COS‐7 and HeLa cells) by the liposomes with derivative I. However, their transfection was completely blocked by nigericin which was able to dissipate the pH gradient across the endosome membranes, although the liposome/DNA complex was found in the cytoplasm of the target cells. This was quite in contrast with the fluorescence images of the target cells treated with wortmannin, an inhibitor of endocytosis. The results suggest that at least two steps are effective for gene transfection mediated by the cationic liposomes with cationic cholesterol derivatives. One is the endocytosis of the liposome/DNA complex into the target cells and the other is the removal of antisense oligonucleotides (plasmid DNAs) from the complex in the endosomes. The latter step was preferentially preceded by the membrane fusion between the cationic liposomes and the endosome membranes at around pH 5.0.

Effect of zeta potential of cationic liposomes containing cationic cholesterol derivatives on gene transfection

Cationic liposomes are known to be useful tools for gene transfection. However, the relation between transfection efficiency and physicochemical properties of liposomes has not been well understood. Here, we synthesized eight cationic derivatives of cholesterol which contain a tertiary amino head group with a different spacer arm. Transfection of plasmid pSV2CAT DNA into cells was done by cationic liposomes made of a mixture of dioleoylphosphatidylethanolamine (DOPE) and each cationic cholesterol derivative. At the same time we measured zeta potential of cationic liposomes by laser Doppler spectroscopy. The present results indicated that zeta potentials of cationic liposomes were well related to transfection activity of pSV2CAT DNA. This suggested that zeta potential of cationic liposomes is one of important factors which control gene transfection.

Atomic force microscopy for studying gene transfection mediated by cationic liposomes with a cationic cholesterol derivative

Atomic force microscopy (AFM) was used for studying gene transfection mediated by cationic liposomes which contain a cationic cholesterol derivative with a different spacer arm. Cationic liposomes were made by a mixture of one of eight cationic cholesterol derivatives and 1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidyl ethanolamine (DOPE). AFM images showed that vesicles made of the liposome/DNA complex had various diameters depending on each cationic cholesterol derivative with a different spacer arm. The results showed that the diameter of the liposome/DNA complex was well related to the transfection activity of plasmid pSV2CAT DNA to a cultured cell line (NIH3T3). From the results it was found that the vesicles with moderate diameters (from 0.4 to 1.4 μm) were moste effective for gene transfection of plasmid pSV2CAT DNA into the target cell. Neither smaller vesicles (<400 nm) nor larger vesicles (>1.4 μm) were adequate for gene transfection. As the gene transfection by the cationic liposomes was mostly inhibited by wortmannin, an inhibitor of endocytosis, it is suggested that the vesicles with moderate diameters were useful for gene transfection by endocytosis.

An in-phantom dosimetry system using pin silicon photodiode radiation sensors for measuring organ doses in x-ray CT and other diagnostic radiology

A dosimetry system using commercially available pin silicon photodiodes as the sensor is evaluated for in-phantom dose measurements in x-ray CT and other diagnostic radiology. System sensitivity measured as a function of the effective energy of x rays was between 0.37 and 0.49 V/mGy at an effective energy range between 23.5 and 72 keV. The minimum detectable organ dose with 25% uncertainty was estimated to be 0.02 mGy. The excellent output linearity was found over a dose range from 0.03 to more than 10 mGy with flat dose rate response of system sensitivity up to 35 mGy s(-1), though the sensitivity indicated some energy dependence across the diagnostic energy range with a maximum of about 10%/10 keV. Since angular dependence of the sensitivity of the photodiode sensor was found to be small enough it would induce negligible dose error. Dose profile measurement along the axis of a thoracic phantom undergoing CT chest examination indicated the reliability of dose values over a range of two orders of magnitude from less than 0.2 to 12 mGy. The present dosimetry system having advantages of high sensitivity with immediate readout of dose values, low cost, and easy construction would widely be used as an alternative to TLD dosimeters for organ and skin dose measurements in CT and other diagnostic radiology.

Evaluation of patient dose for barium enemas and CT colonography in Japan

The objective of this study was to assess patient doses for examinations of the lower digestive tract (barium enemas and CT colonography) in Japan. These doses were evaluated from in-phantom dose measurements using a dosemeter-implanted anthropomorphic phantom and from the knowledge of procedures of these examinations. For barium enemas, the doses, which were the sums of doses for various projections in the procedure, were separately derived for fluoroscopy and for analogue and digital radiography. For CT colonography, the doses were evaluated for the prone and the supine positions, each including the doses by scout imaging, and a single abdominal scan for routine and low-dose set-ups. For barium enemas, maximum local skin doses were less than 100 mGy despite relatively long average fluoroscopy times of 8 min; organ doses ranged from 9-26 mGy in the abdomen. The effective dose of 10.7 mSv for analogue radiography decreased by 12% when digital radiography was used, although more than 80% of the dose was due to fluoroscopy. In routine CT colonography performed using a relatively high mean effective mAs of 119 for the accurate detection of colorectal cancer and extra colonic lesions, organ doses within the primary X-ray beam were between 30 mGy and 44 mGy for paired scans whereas, in a low-dose set-up with an effective mAs of 27, they were approximately 10 mGy. Effective doses for routine and low-dose CT colonography of 23.4 mSv and 5.7 mSv were about double and half of the doses for barium enemas, respectively.

Dose reduction and image quality in CT angiography for cerebral aneurysm with various tube potentials and current settings

OBJECTIVES The purpose of this study is to investigate the image quality on both axial and three-dimensional CT angiograms of the brain at various tube potentials and currents, and to propose the use of descriptors for evaluating the image quality of three-dimensional CT angiograms using entropy analysis. METHODS A head phantom was used as a target object. Axial CT and three-dimensional CT angiograms were obtained at various effective milliampere-second values (49-350 mAs) and tube potentials (80-140 kVp) with a 64-row detector CT scanner. Lens doses were measured using a planar silicon pin-photodiode system. The signal-to-noise ratio (SNR) and streak artefacts on the axial CT angiograms were evaluated and the image quality of the three-dimensional CT angiograms was assessed using entropy analysis. RESULTS Lens doses increased with tube potential and effective milliampere-seconds. From the evaluation of SNR and streak artefacts on axial CT angiograms, we found that the image quality was improved by setting the tube potential at 100 kVp. However, there was little visual difference in the image quality for 100 kVp between 252 (effective value recommended by the manufacturer) and 350 mAs (maximum effective value). In the entropy analysis of the image quality of three-dimensional CT angiograms, the mutual information (information gain) per lens dose was largest at 80 kVp and 252 mAs. CONCLUSION Our results suggested that the suitable tube potentials for axial CT and three-dimensional CT angiograms were 100 and 80 kVp, respectively, and the effective milliampere-second value recommended by the manufacturer was appropriate.

Contrast enhancement efficacy of iodinated contrast media: Effect of molecular structure on contrast enhancement

Highlights • We derived a new formula for comparing the contrast enhancement efficacy.• Theoretical value agrees with actual value for each combination of contrast media.• The contrast enhancement is affected by molecular structure of contrast medium.